| The outbreak of Sever Acute Respiratory Syndrome (SARS) caused by SARS Coronavirus (SARS-CoV ) during the spring of 2003 had brought great attention worldwide. Early, rapid and accurate diagnostic approaches are very important for quarantine of the source of infection. Currently, laboratory examinations were mainly immunological as exemplified by ELISA, colloidal gold indicator paper etc. PCR as well as the conventional virus identification method by tissue culture had also been employed. These methods, though practical, prove to be less optimal for early diagnosis. Oligonucleotide microarray technology was applied in this study in an attempt to find an early, rapid, accurate, sensitive and more efficient method for SARS-CoV detection.DNA Microarray is a new developed technology for gene detection in recent years. Especially the progress of resolvent applied now days ,such as the 60~70mer long oligonucleotide Microarray, had provided a possibility for high specific detection of SARS-CoV. In this research, we also adopted a whole genome labeling method to match the high throughput characteristic of microarray diagnosis, so as to contribute to the application of microarray technology in clinical diagnosis.To begin with, an optimal condition for long oligonucleotide microarray experiment was established. Several substrates were applied to achieve a satisfactory immobilization ratio and different probe concentrations were tested to got the most appropriate one for printing. Furthermore, length of the probes for the best immobilization ratio and cost efficiency were determined for the preparation of oligo microarray, for which 60 mer oligos were found more optimal for this study.Sequences of SARS-CoV were analyzed with the NCBI GENEBANK database, 60mer oligonucleotide probes were designed by bioinformatics methods and aligned by BLAST to eliminate the sequences that were notspecifics to the SARS-CoV. Thirty oligonucleotides were originally selected and synthesized, to printing into an oligo microarray for SARS-CoV detection.Virus RNAs extracted from SARS-CoV in vero E6 cell culture was used as positive controls, extracted total RNAs of Homo sapiens were used as negative controls to compare with. RNA samples were labeled by restriction labeling method and were applied to the microarray for hybridization. The results were scanned by a confocal scanner. The images were extracted by Array-Pro Analyzer software, data collected were compared to evaluate the probes and for the final selection of candidate probes for further oligo microarray preparations.After the probe selection had been completed, the application of this microarray to clinical diagnosis was studied. To resolve the problem of low virus titer of clinical samples and to avoid possible contaminations, the restriction labeling method was used and enhanced by adding a fluorescent labeled dNTP into the labeling reaction. The fluorescent intensity per sample molecular could be increased which boost the sensitivity of the diagnosis. Processes for avoiding contamination from the amplified production were adopted so that false positive result eliminated effectively.The optimized microarray and microarray kit were applied for pilotscale clinical experiments. Six hurand and one samples were collected by Center for Diease Control of Guang Dong province, among which 200 saliva samples were from patients been diagnosed as SARS, while 401 saliva samples were from normal people or non-SARS subjects. All of the samples had been treated double blindly, clinical data analysis method and diagnostic standard had been established to evaluate the result of this microarray kit for SARS-CoV detection. Through experimental study, we reached the following conclusions:1. It has been found that poly-L-lysine treated slides had the highestimmobilization ratio among all kind of reagents for coating. The immobilization ratio increased with the concentration of the probes when it is between 1.0-2.5 n g/ u L, these would be the best choice for probe concentration of an olig...
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